Superoxide dismutase mutant and application thereof

Abstract

The invention discloses superoxide dismutase mutants and application thereof.The superoxide dismutase mutants are E47S, G109H and D120L mutants of superoxide dismutase shown in SEQ ID NO: 1, the enzyme activities of the three mutants are all improved compared with original enzymes, the activity of the G109H mutant is improved by 145.9%, the thermal stability of the E47S mutant is improved by 22.5%, the industrial application value of SOD is further improved, and the superoxide dismutase mutant has the advantages that the superoxide dismutase mutant is suitable for industrial production. The method can be widely applied to the fields of cosmetics, food, medicine, environmental protection and the like.

Description

Technical Field

[0001] The invention relates to the field of biological genes, and in particular to a superoxide dismutase mutant and application thereof. Background Art

[0002] Superoxide dismutase (SOD) is an important antioxidant enzyme in organisms, widely distributed in various organisms, such as animals, plants, microorganisms, etc. SOD has special physiological activity and is the primary substance for removing free radicals in organisms. The level of SOD in organisms is a direct indicator of aging and death. It has been confirmed that there are more than 60 diseases caused by oxygen free radicals. It can resist and block the damage caused to cells by oxygen free radicals, and repair damaged cells in time to restore cell damage caused by free radicals. Due to the stress of modern life, environmental pollution, various radiations and excessive exercise, a large number of oxygen free radicals will be formed. Therefore, the position of SOD in the biological antioxidant mechanism is becoming more and more important.

[0003] Superoxide dismutase can be divided into three types according to the different metal cofactors it contains. The first type is SOD containing copper and zinc metal cofactors, called Cu.Zn-SOD, which is the most common SOD enzyme, green in color, and mainly exists in the cytoplasm of the body; the second type is SOD containing manganese metal cofactors, called Mn-SOD, purple in color, and exists in the mitochondria of eukaryotic cells and prokaryotic cells; the third type is SOD containing iron metal cofactors, called Fe-SOD, yellow-brown in color, and exists in prokaryotic cells.

[0004] SOD has the functions of anti-aging, immune regulation, blood lipid regulation, radiation resistance, beauty and so on, and has important uses in the fields of cosmetics, food, medicine, environmental protection, etc. However, the SOD in the prior art has defects in heat resistance and stability, which greatly limits the application of SOD.

[0005] The main methods for producing SOD are natural extraction (mainly plant and animal tissue) and microbial fermentation. Currently, due to resource limitations, unstable supply chains, ethical issues, etc., microbial fermentation has developed rapidly. Among them, the use of genetically engineered microorganisms has made it possible to stabilize the production process, reduce production costs, and expand the experimental process.

[0006] There is a growing body of research on the microbial production of SOD at home and abroad, but one of the key issues is that the SOD produced cannot meet the needs of practical applications. Limited thermal stability and catalytic efficiency remain important obstacles to the wider industrial application of SOD. For example, the SOD enzyme from Lactobacillus thermocyclus (Frontiers in Microbiology 12 (2021): 577001) retains only 30% of its activity after being kept at 80°C for 60 minutes. Summary of the invention

[0007] The first object of the present invention is to overcome the deficiencies of the prior art and provide a superoxide dismutase mutant having both good heat resistance and activity.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] A superoxide dismutase mutant, wherein the 47th amino acid residue of superoxide dismutase is mutated to serine;

[0010] or mutating the 109th amino acid residue of superoxide dismutase to histidine;

[0011] Or the 120th amino acid residue of superoxide dismutase is mutated to leucine;

[0012] Wherein, the amino acid sequence of the superoxide dismutase is shown as SEQ ID NO:1.

[0013] The second object of the present invention is to provide a gene encoding the above-mentioned superoxide dismutase mutant.

[0014] The third object of the present invention is to provide a recombinant vector comprising the above gene. Preferably, the gene is cloned into a pET-22b plasmid to construct the recombinant vector.

[0015] The fourth object of the present invention is to provide a recombinant strain containing the above gene. Preferably, the recombinant strain uses Escherichia coli as a host bacterium.

[0016] The fifth object of the present invention is to provide a method for expressing the mutant, specifically, using an E. coli expression system to express the enzyme protein.

[0017] As a preferred embodiment, the expression method comprises: cloning the gene sequence of the mutant into a vector plasmid, constructing a recombinant expression vector, and transferring the recombinant expression vector into Escherichia coli for induced expression.

[0018] As a preferred embodiment, the recombinant Escherichia coli culture solution was added to LB medium and cultured at 37°C and 200 rpm until the culture solution OD600 = 0.3~0.5, add IPTG inducer to induce expression

[0019] The seventh object of the present invention is to provide the use of the above mutant in the preparation of antioxidant products. The superoxide dismutase mutant can resist and block the damage to cells caused by oxygen free radicals generated by environmental pollution, various radiations and excessive exercise, and repair damaged cells in time. It can be applied to cosmetics, fruit and vegetable preservation, and pharmaceutical products.

[0020] The present invention purifies and expresses the SOD enzyme derived from Urumqi Deinococcus R12 (abbreviated as R12-SOD), and mutates the SOD enzyme through semi-rational enzyme engineering transformation technologies such as site-directed mutagenesis, obtaining three mutants with improved thermal stability and activity, namely E47S, G109H, and D120L. The enzyme activities of the three mutants are all improved compared with the original enzymes, among which the activity of the G109H mutant is improved by 145.9%, and the thermal stability of the E47S mutant is improved by 22.5%, further improving the industrial application value of SOD, which can be widely used in cosmetics, food, medicine, environmental protection and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the thermal stability test results of R12-SOD in Example 2 at 80°C, 90°C, and 100°C.

[0022] Figure 2 This is a graph showing the results of the enzyme activity test of the wild type R12-SOD and three mutants in Example 4.

[0023] Figure 3 This is a graph showing the thermal stability test results of the wild type and three mutants of R12-SOD in Example 4 after incubation at 100°C for 60 minutes. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The experimental methods without specific conditions are based on conventional experimental conditions in the art or conditions recommended by the manufacturer.

[0025] The SOD gene of the present invention is derived from Deinococcus wulumuqiensis R12, and its amino acid sequence is shown in SEQ ID NO:1.

[0026] Example 1

[0027] The protein expression vector of SOD gene was constructed through molecular cloning technology.

[0028] 1. Codon optimization and plasmid construction

[0029] The nucleotide sequence of the Mn-SOD gene encoded by Deinococcus urumqiensis-R12 was codon-optimized, artificially synthesized after adding a His-tag at the 5' end, and connected to the pET-22b vector (completed by General Motors) through the cloning site NdeI-BlpI.

[0030] 2. Conversion

[0031] Take out the E. coli BL21 competent cells and plasmid from the -80°C and -20°C freezers, respectively, and thaw them on ice.

[0032] Add 100µL of thawed competent cells and 10µL of plasmid into the tube, stir gently with the pipette tip, and keep on ice for 30 minutes.

[0033] Heat stimulate at 42°C for 90 seconds and quickly return to ice for 2 minutes.

[0034] Add 700 µL of LB liquid medium without antibiotics to the competent cells containing the plasmid and incubate on a shaker at 37°C for 1 h.

[0035] Take 100 µL of bacterial solution and spread it on LB solid medium containing corresponding antibiotics.

[0036] The culture dish was inverted and incubated at 37°C overnight.

[0037] 3. Sequencing verification and construction of expression strains

[0038] Multiple single colonies were selected from the culture dishes cultured overnight after transformation, inoculated into liquid LB medium with ampicillin resistance, cultured overnight, and verified by bacterial liquid sequencing. Sequencing was performed using T7 upstream and downstream universal sequencing primers, and the results showed that the sequence was correct.

[0039] Example 2

[0040] Protein induction expression, activity and thermal stability test of R12-SOD

[0041] 1. Inducible expression

[0042] Take 300 μL of the bacterial solution with the correct sequence and cultured overnight in step 3 of Example 1, add it to 50 mL of LB medium, add ampicillin, and culture at 37°C and 200 rpm.

[0043] When the OD value reached 0.5 (0.3-0.5), IPTG was added to a final concentration of 0.1 mM, and then cultured at 37°C, 200 rpm for 12 h.

[0044] 2. Ultrasonic cell disruption (30 mL bacterial solution)

[0045] Pour 30mL of bacterial solution into a 50mL centrifuge tube, balance it, centrifuge it at 8000rpm for 5min, discard the supernatant, and continue to add bacterial solution to the centrifuge tube. Balance it with RO water, continue centrifugation, discard the supernatant, and finally add 5mL of PBS to resuspend it by pipetting, centrifuge it, and discard the supernatant.

[0046] After all the cells are collected, add 2 mL of PBS to dissolve all the bacterial masses and transfer them to the corresponding 5 mL centrifuge tube. Then add 1 mL of PBS to clean the centrifuge tube and pour it into the 5 mL centrifuge tube.

[0047] Ultrasonic disruption was performed in an ice bath with an ultrasonic power of 300 W, and the disruption lasted for 3 s followed by a 5 s break for a total of 10 min.

[0048] The disrupted bacterial solution was centrifuged at 4°C, 5000 rpm, for 20 min.

[0049] The supernatant and the precipitate were separated for later use. The precipitate was resuspended in 2 mL of PBS and then sampled for subsequent protein gel running. The supernatant was used for purification and stored in a -20°C refrigerator.

[0050] The protein sequence of the SOD expressed by genetic engineering recombinant in the supernatant is shown in SEQ ID NO:1.

[0051] 3. Protein purification

[0052] Drain the alcohol from the nickel column stored in -20℃ alcohol, and add 5mL 30 Mm imidazole buffer (BufferA), 3mL 300 Mm imidazole buffer (BufferB), and 5mL BufferA to wash the column.

[0053] Add crude enzyme solution and wash twice with 5 mL of Buffer A to remove impurities. Then add 3 mL of Buffer B and pour into the prepared labeled beaker to obtain the target protein.

[0054] Add 3 mL Buffer B and 5 mL Buffer A to wash the column and seal it with 20% alcohol.

[0055] Pour the crude enzyme solution in the beaker into an ultrafiltration tube (sealed with ultrapure water) and centrifuge at -4°C and 6500 rpm for 30 min.

[0056] After completion, add 2 mL of ultrapure water and centrifuge at 6500 rpm for 30 min.

[0057] Add 2 mL of ultrapure water and centrifuge at 6500 rpm for 15 min to obtain the concentrated pure enzyme solution. Store the concentrated pure enzyme solution in a -4°C refrigerator.

[0058] 4. Activity detection

[0059] At about 25℃, add 94 μL of 0.1mol / L Tris-HCL (1mmol / L EDTA•2Na), 80 uL of 1× PBS buffer, and 6 uL of 4.5mmol / L pyrogallol hydrochloric acid solution to a 96-well plate colorimetric tube. Immediately after adding 4.5mmol / L pyrogallol hydrochloric acid solution, mix well and pour into the colorimetric dish, measure the absorbance values ​​at 0, 30s, 60s, 90s, 120s, 150s, and 180s at a wavelength of 325nm, and calculate the absorbance increment from 30s to 150s, that is, the absorbance value of 150s minus the absorbance value of 30s divided by 2 to obtain the pyrogallol self-oxidation rate ΔA325 (min-1).

[0060] The sample solution was diluted to 0.05 mg / mL. The inhibition rate of pyrogallol self-oxidation by the sample solution was determined by adding a certain amount of sample solution according to step 1 so that the inhibition rate of pyrogallol self-oxidation was about 1 / 2ΔA325 (min-1), that is, ΔA'325 (min-1).

[0061] Total enzyme activity =

[0062] Specific enzyme activity = total enzyme activity

[0063] The test results showed that the specific enzyme activity of R12-SOD was 1351U / mg.

[0064] 5. Thermal stability test

[0065] The pure enzyme solution was kept at 80°C, 90°C, and 100°C, and the SOD enzyme activity was measured at 15 minutes, 30 minutes, 45 minutes, and 60 minutes, respectively. The thermal stability was calculated and expressed as the activity retention percentage.

[0066] The results are as follows Figure 1 As shown:

[0067] The SOD enzyme of the present invention still retains 76% of its activity after being incubated at 80°C for 60 minutes, and still retains 73% of its activity after being incubated at 90°C for 60 minutes, and still retains 48% of its residual activity even after being incubated at 100°C for 60 minutes, indicating that the SOD enzyme of the present invention has good thermal stability.

[0068] Example 3

[0069] Site-directed mutagenesis of R12-SOD superoxide dismutase

[0070] The 47th glutamic acid, the 109th glycine, and the 120th aspartic acid in the amino acid sequence of the SOD enzyme shown in SEQ ID NO: 1 were mutated to serine, histidine, and leucine, respectively, to obtain three mutants, E47S, G109H, and D120L. Flat-end primers were designed using SnapGene 6.0.2, and PCR site-directed mutagenesis was performed using the R12-SOD enzyme of the present invention as a template.

[0071] The base sequences of the primers are shown in Table 1:

[0072] Table 1 Primer sequences

[0073] The PCR reaction system and cycle process are shown in Table 2 and Table 3 respectively:

[0074] Table 2 PCR reaction system

[0075] Table 3 PCR cycle process

[0076] After the PCR reaction, 1 μL of DpnI and 5 μL of 10×EC Buffer were added to the 50 μL system, and the mixture was incubated at 37°C for 1 h 30 min, and then at 80°C for 45 min to digest the template. Agarose gel electrophoresis was used to detect whether the site-directed mutation was completed. After successful verification, the PCR product was recovered, and the gene was cloned into an expression vector according to the methods of Examples 1 and 2, and the target protein was induced to be expressed and purified.

[0077] 1. Activity detection

[0078] The specific enzyme activities of E47S, G109H, and D120L mutants were measured using the pyrogallol method.

[0079] like Figure 2 As shown, the specific enzyme activities of E47S, G109H, and D120L mutants were 1726.9 U / mg, 3322.5 U / mg, and 1442.1 U / mg, respectively.

[0080] 2. Thermal stability test

[0081] The pure enzyme solution was kept at 100°C, and the SOD enzyme activity was measured at 15 minutes, 30 minutes, 45 minutes and 60 minutes, and its thermal stability was calculated and expressed as the percentage of activity retention. Figure 3The results showed that the residual activities of the three mutants E47S, G109H, and D120L were 89.4%, 48.2%, and 87.2%, respectively, after incubation at 100°C for 60 min. The specific enzyme activities of the wild type and the three mutants E47S, G109H, and D120L and the thermal stability data after incubation at 100°C for 60 min are shown in Table 4.

[0082] Table 4 Activity and thermal stability of the wild type and three mutants: E47S, G109H, and D120L

[0083] The results of the wild-type superoxide dismutase assay in Example 2 showed that the specific enzyme activity and thermal stability of the three mutants E47S, G109H, and D120L were improved compared to the wild type, among which the specific enzyme activity of the G109H mutant reached 3322.5 U / mg, an increase of 145.9%, and the E47S mutant still retained 89.4% of its activity after incubation at 100°C for 60 minutes, an increase of 22.5%. The improvement of the activity and thermal stability of the mutants further broadened the industrial application value of SOD.

Claims

1. A superoxide dismutase mutant, characterized in that: The amino acid residue 47 of superoxide dismutase was mutated to serine; or mutating the 109th amino acid residue of superoxide dismutase to histidine; Or the 120th amino acid residue of superoxide dismutase is mutated to leucine; Wherein, the amino acid sequence of the superoxide dismutase is shown as SEQ ID NO:

1.

2. A gene encoding the superoxide dismutase mutant according to claim 1.

3. A recombinant vector comprising the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The gene was cloned into pET-22b plasmid to construct the recombinant vector.

5. A recombinant strain comprising the gene according to claim 2.

6. The recombinant strain according to claim 5, characterized in that The recombinant strain uses Escherichia coli as a host bacterium.

7. The method for expressing the mutant according to claim 1, characterized in that: The enzyme protein was expressed using the E. coli expression system.

8. The expression method according to claim 7, characterized in that: The gene sequence of the mutant is cloned into a vector plasmid to construct a recombinant expression vector, which is then transferred into Escherichia coli for induction of expression and purification.

9. The expression method according to claim 8, characterized in that The induction expression conditions were as follows: the recombinant E. coli culture was added to LB medium and cultured at 37°C and 200 rpm until the culture OD reached 600 =0.3~0.5, add IPTG inducer to induce expression.

10. Use of the mutant according to claim 1 in preparing antioxidant products.

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